The present invention relates generally to the virulence of pathogenic organisms and more specifically to virulence factors associated with enteropathogenic bacteria.
Antibiotics have been used for years to successfully treat diverse bacterial infections. However, bacterial resistance to antibiotics has been an increasing problem over the past few years. Many pathogens are now resistant to several antibiotics, and in some cases, the diseases they cause are no longer treatable with conventional antibiotics. Despite the past successes of antibiotics, there have been few, if any, new classes of antibiotics developed in the past two decades. New variations on existing drugs have been introduced, but resistance to these compounds usually arises within a short period of time.
Many studies have shown that if a mutation is made in a gene that encodes a virulence factor, the organism containing that gene is no longer pathogenic. Additionally, if a host is vaccinated against a virulence factor, disease can often be blocked. However, it has not been shown that specific inhibition of a virulence factor can attenuate disease.
The mechanisms of action for toxins, adherence, invasion, intracellular parasitism, have been studied. However, each virulence factor uses a different mechanism, which has made the development of a broad spectrum inhibitor impossible. One conserved factor that could be considered a target for a therapeutic is a two-component regulatory system. However, this system is not specific for virulence factors, and is used in several bacterial housekeeping systems. Additionally, the systems have been identified in eukaryotic systems, which would increase the risk of host toxicity if an inhibitor was utilized. To develop an ideal anti-infective agent, the bacterial virulence mechanism that the antibiotic affects should be universal for many pathogens, specific for virulence mechanisms, and not be present in host cells. One such system that has recently been identified is the bacterial type III secretion system.
Gram-negative bacteria utilize specialized machinery to export molecules across their two membranes and the piroplasm, a process critical for moving virulence factors to the bacterial surface where they can interact with host components. Gram-negative secretion has been divided into four major pathways. First, the Type I secretion is used by a small family of toxins, with E. coli hemolysin being the prototype. Second, the type II secretion system is the major export pathway used by most Gram-negative bacteria to export many molecules, including some virulence factors; it shares homology to mammalian drug resistance mechanisms. Third, the type IV secretion system is encoded within the secreted product, which cleaves itself as part of the secretion mechanism; the prototype of this system is the Neisseria IgA protease. Fourth, the most recently discovered secretion pathway, is the type III pathway.
Type III secretion systems were originally described as a secretion system for Yersinia secreted virulence proteins, YOPs, which are critical for Yersinia virulence. A homologous secretion system was then identified in several plant pathogens, including Pseudomonas syringae, P. solanacearurn, and Xantharnonas carnpestris. These plant pathogens use this secretion pathway to secrete virulence factors (harpins and others) that are required for causing disease in plants. Although the secretion system is similar, harpins and YOPs (i.e. the secreted virulence factors) are not homologous polypeptides. Several other type III secretion systems necessary for virulence have more recently been identified in other pathogens. These systems include the invasion systems Salmonella and Shigella use to enter cells and cause disease. Another type III secretion system has been identified in Salmonella which is critical for disease, although the secreted products of this pathway and the virulence mechanisms have not been established yet. Pseudomonas aeruginosa has a type III secretion system necessary for secretion of Exoenzyme S, a potent virulence factor.
Enteropathogenic Escherichia coli (EPEC) is a leading cause of infant diarrhea and was the first E. coli shown to cause gastroenteritis. Enteropathogenic E. coli activates the host epithelial cells"" signal transduction pathways and causes cytoskeletal rearrangement, along with pedestal and attaching/effacing lesion formation.
A three-stage model has describes enteropathogenic E. coli pathogenesis. An initial localized adherence to epithelial cells, mediated by a type IV fimbria, is followed by the activation of host epithelial cell signal transduction pathways and intimate attachment to host epithelial cells. These final two steps are collectively known as attaching and effacing. The signal transduction in the host epithelial cells involves activation of host cell tyrosine kinase activity leading to tyrosine phosphorylation of a 90 kilodalton host membrane protein, Hp90, and fluxes of intracellular inositol phosphate (IP3) and calcium. Following this signal transduction, the bacteria adheres intimately to the surface of the epithelial cell, accompanied by damage to host epithelial cell microvilli and accumulation of cytoskeletal proteins beneath the bacteria.
The present invention is based on the discovery a protein associated with virulence in pathogenic bacteria, for example enteropathogenic E. coli. 
DNA sequence analysis of the Locus of Enterocyte Effacement between eaeA and espB identified a gene (espA) that matched the amino terminal sequence of the 25 kilodalton enteropathogenic E. coli secreted protein. A mutant with an insertion in espA does not secrete this protein, activate epithelial cell signal transduction or cause cytoskeletal rearrangement. However, these functions could be complemented by a cloned wild-type espA gene.
Two enteropathogenic E. coli genes, espA and espB, that encode secreted virulence factors, EspA and EspB respectively, were cloned and sequenced. These proteins were shown to be involved in the triggering of host epithelial signal transduction pathways and invasion. Since EspA is a secreted protein, it is ideally suited for use in a fusion protein linked to a polypeptide of interest.
The type III secretion pathway is an ideal target for potential inhibitors, because it is a virulence factor-specific conserved pathway identified in bacteria. The present invention provides a method for identifying inhibitors of type III secretion systems. The relevance of this invention is directed toward the development of new antibacterial therapeutics. In contrast to other antibiotics, the compounds identified by the method of this invention will not kill or inhibit growth of pathogens; instead, the compounds will block the secretion of virulence factors that are critical to causing disease. Because the type III secretion system is the first virulence mechanism that shows a large degree of conservation between diverse pathogens, some of the compounds identified by the method of this invention will be broad spectrum therapeutics. The benefit would be the identification of new therapeutics that could be used in the treatment of several human, animal, and plant diseases.